KRAS is a Kirsten ras oncogene homolog from the mammalian ras gene family, which encodes a protein that is a member of the small GTPase superfamily. Mutations in the KRAS oncogene are frequently found in human pathologies, in particular cancer, wherein a single amino acid substitution in the KRAS protein may be responsible for an activating mutation. The mutated protein that results may be implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma.
Also, defects in KRAS can be a cause of acute myelogenous leukemia (AML), of juvenile myelomonocytic leukemia (JMML), of Noonan syndrome type 3 (NS3), and of cardiofaciocutaneous syndrome (CFC syndrome). Nearly 50% of colon cancers harbour activating mutations in KRAS, and also activating mutations in the KRAS oncogene are commonly associated with progression from a benign adenoma to an advanced/dysplastic adenocarcinoma. In addition, the presence of these mutations in KRAS correlates with a lack of response to certain anti-cancer therapies based on EGRF inhibition, in metastatic colorectal cancer patients. The evaluation of KRAS mutational status is thus often recommended to determine appropriate treatment.
Neoplasia-associated KRAS mutations frequently affect codons 12, 13 and 61. Mutations in codons 12 and 13 of KRAS are strong predictors of non-response to anti-EGFR antibodies in metastatic colorectal cancer (Shankaran et al., 2010, Oncologist 15, 157-167). Also, clinical studies indicate that codon 61 mutations are also associated with poor outcomes from anti-EGFR antibody therapy (De Roock et al., 2010, Lancet Oncol. 11, 753-762; Loupakis et al., 2009, Br. J. Cancer 101, 715-721). Codons 12 and 13 of the KRAS oncogene harbour 7 relevant mutations of KRAS: Gly12Ser (GGT/AGT), Gly12Arg (GGT/CGT), Gly12Cys (GGT/TGT), Gly12Asp (GGT/GAT), Gly12Ala (GGT/GCT), Gly12Val (GGT/GTT), and Gly13Asp (GGC/GAC). Also, codon 61 mutations, in particular, Gln61His (CAA>CAT) and Gln61Leu (CAA>CTA) are missense mutations, which abolish GTPase activity resulting in constitutively activated ras signaling.
There is a high demand for methods of detection of these 9 mutations of the KRAS gene. In particular, the state of the art often deals with the issue of detecting at least the 7 mutations corresponding to Gly12 and Gly13. FIG. 1a) displays the DNA fragment that contains the positions that give rise to the 7 KRAS mutations Gly12Ser, Gly12Arg, Gly12Cys, Gly12Asp, Gly12Ala, Gly12Val and Gly13Asp (the 5 nucleotides that determine the exact KRAS mutation are contained within the square). FIG. 1b) shows the exact nucleotide changes that give rise to each of the 7 KRAS mutations. A consequence of the fact that the 7 KRAS mutations all lay in close proximity within the KRAS gene sequence, and of the minimal sequence differences between the corresponding amplification products, is the non-specific hybridisation of any amplification product corresponding to one of the KRAS mutations, with probes complementary to the other KRAS mutations. Thus, hybridization to a probe of the DNA amplification product of any of the 7 KRAS mutations, is not specific, as the amplification fragment of other KRAS mutations may also non-specifically bind to the probe corresponding to the first one.
This drawback applies both to multiplex ARMS amplification, as well as to the individual ARMS amplification approach, wherein specific amplification products are obtained from each mutation in independent reaction vessels.
Another associated problem is that the KRAS mutations that may present as prognostic factors for tumour staging, metastasis, evolution, cellular heterogeneity, or allelic heterogeneity, are often to be found in samples in low abundance with respect to the wild type form. And, although many diagnostic methods are available for mutation detection, most cannot accurately detect low-abundance mutations. Sanger sequencing is the gold standard for KRAS mutation identification though it may only detect mutations in abundances above approximately 20%.
US2003/175750A1 (Barany Francis (US) et al.), discloses a method for detection of one or more nucleic acid differences, in particular, KRAS mutations, the method comprising a ligation step between two oligonucleotide probes (“Ligation Detection Reaction” or “LDR”) as crucial step. According to the information displayed in US2003/175750A1, LDR would be the detection method for closely-clustered mutations such as those of KRAS, which are not amenable to detection by allele-specific PCR or hybridization.
Some other known detection methods used in the state of the art are based on PCR amplification of the DNA fragment of KRAS containing these mutations. The high sequence similarity between the DNA sequences of the KRAS mutations, prejudices specificity of detection of the amplification products. Subsequent detection of the PCR products in most methods of the state of the art thus takes place through some technical approaches such as DNA sequencing, visualisation in agarose gel, etc.
Two particular documents of the state of the art, WO 99/04037 and WO 2010/048691 disclose diagnostic methods for the detection of KRAS mutations, based on the amplification refractory mutation system (ARMS). ARMS is a method of detecting point mutations, based on the principle of allele-specific priming of PCR amplification (EP0332435; Newton et al., 1989, Nucleic Acid Research 17, 2503-2516). This system is based on a strategy wherein an oligonucleotide primer is designed so that it only functions as a primer for the PCR when it anneals to its specific target DNA sequence. The technique requires that the terminal 3′-nucleotide of the PCR primer be allele specific. This implies that the terminal 3′-nucleotide corresponds to that of the point mutation. Thus, the primer is designed in two forms: The “normal” form, which is refractory to PCR to “mutant” template DNA, and the “mutant” form, which is refractory to PCR on “normal” DNA.
In some instances, a single 3′-mismatched base does not completely prevent the non-specific extension of the oligonucleotide primer when having as target the DNA corresponding to another point mutation, and amplification proceeds.
In such cases, introduction of a deliberate mismatch near the 3′ end of the allele-specific appropriate primer (at the second, third, or even fourth nucleotide from the 3′ end of the primer) allows to enhance the specificity of the primer.
The ARMS technique involves that at least two PCRs may take place in one reaction mixture, each corresponding to amplification with one of the ARMS primers. Any ARMS primer further requires a second primer (that will be hereafter called amplification primer, and which is usually also called the common primer) to generate the allele-specific product. In addition, two or more control primers may be included in the reaction mixture in order to generate an unrelated product that indicates that the reaction is working correctly.
WO 99/04037 discloses detection of the 7 KRAS mutations Gly12Ser, Gly12Arg, Gly12Cys, Gly12Asp, Gly12Ala, Gly12Val and Gly13Asp present in a sample, based on ARMS amplification with the ARMS primers 1 to 7 displayed in Table 2 of WO 99/04037. Further to WO 99/04037, Yamada et al., 2005, Int. J. Cancer 113, 1015-1021, makes use of individual PCR amplification reactions, specific for each mutant allele, for detecting KRAS mutations in tumour and normal colorectal mucosa samples. Visualization of the amplification products is carried out after agarose gel electrophoresis and ethidium bromide staining. Simultaneous detection of ARMS products corresponding to different KRAS mutations, through hybridization with detection probes, for instance, would not be possible, the reason being the non-specific binding of the ARMS products of certain KRAS mutations to the probes of different KRAS mutations, due to sequence similarity.
WO 2010/048691 discloses a method of detection of KRAS mutations also based in ARMS amplification, but wherein the ARMS primers display a 3′ fragment of 19 to 21 nucleotides (nt), complementary to the target sequence of the KRAS mutation to be detected, and a different non-specific 5′ sequence that is used for detection. Thermal cycling conditions that are used for DNA amplification comprise a “hybridization temperature” of 54° C. and, usually, 50 PCR cycles are necessary for sensitive amplification. The 5′ non-specific sequence is then used for detection of the amplification products. Detection of 50%, 25% or 5% mutant in wt background was tested with this method, detection of 5% mutant present in a wt background being the best sensitivity data achieved, but not for all KRAS mutations.
A drawback of this method is that some specificity problems arise, as it can be observed in data corresponding to Q61R, in Tables 8 and 9 of the patent application. Basically, not only the KRAS mutation present in the sample, but also other KRAS mutations, not present therein, are detected upon amplification with the ARMS primers of WO 2010/048691.
It would therefore be highly desirable to provide for an alternative method of detection of KRAS mutations present in a sample, that would allow specific detection of such mutations, while maintaining or enhancing the sensitivity values of the methods of the state of the art, in particular, of WO 2010/048691. The method should thus allow detection of KRAS mutations present within the sample in a 5% percentage or lower, in a wt background.
The invention described herein is aimed at providing a robust and reliable method for detecting KRAS mutations, and thus at mitigating the shortcomings in the prior art.